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Coordinated cell movement is a cardinal feature in tissue organization that highlights the importance of cells working together as a collective unit. Disruptions to this synchronization can have far-reaching pathological consequences, ranging from developmental disorders to tissue repair impairment. Herein, it is shown that metal oxide nanoparticles (NPs), even at low and non-toxic doses (1 and 10 µg mL -1 ), can perturb the coordinated epithelial cell rotation (CECR) in micropatterned human epithelial cell clusters via distinct nanoparticle-specific mechanisms. Zinc oxide (ZnO) NPs are found to induce significant levels of intracellular reactive oxygen species (ROS) to promote mitogenic activity. Generation of a new localized force field through changes in the cytoskeleton organization and an increase in cell density leads to the arrest of CECR. Conversely, epithelial cell clusters exposed to titanium dioxide (TiO 2 ) NPs maintain their CECR directionality but display suppressed rotational speed in an autophagy-dependent manner. Thus, these findings reveal that nanoparticles can actively hijack the nano-adaptive responses of epithelial cells to disrupt the fundamental mechanics of cooperation and communication in a collective setting., (© 2024 Wiley‐VCH GmbH.)

Neurofibromatosis type 1, a genetic disorder caused by pathogenic germline variations in NF1, predisposes individuals to the development of tumors, including cutaneous and plexiform neurofibromas (CNs and PNs), optic gliomas, astrocytomas, juvenile myelomonocytic leukemia, high-grade gliomas and malignant peripheral nerve sheath tumors (MPNSTs), which are chemotherapy- and radiation-resistant sarcomas with poor survival. Loss of NF1 also occurs in sporadic tumors, such as glioblastoma (GBM), melanoma, breast, ovarian and lung cancers. We performed a high-throughput screen for compounds that were synthetic lethal with NF1 loss, which identified several leads, including the small molecule Y102. Treatment of cells with Y102 perturbed autophagy, mitophagy and lysosome positioning in NF1-deficient cells. A dual proteomics approach identified BLOC-one-related complex (BORC), which is required for lysosome positioning and trafficking, as a potential target of Y102. Knockdown of a BORC subunit using siRNA recapitulated the phenotypes observed with Y102 treatment. Our findings demonstrate that BORC might be a promising therapeutic target for NF1-deficient tumors., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

There is a paradigm shift in biomanufacturing toward continuous bioprocessing but cell-based manufacturing using adherent and suspension cultures, including microcarriers, hydrogel microparticles, and 3D cell aggregates, remains challenging due to the lack of efficient in-line bioprocess monitoring and cell harvesting tools. Herein, a novel label-free microfluidic platform for high throughput (≈50 particles/sec) impedance bioanalysis of biomass, cell viability, and stem cell differentiation at single particle resolution is reported. The device is integrated with a real-time piezo-actuated particle sorter based on user-defined multi-frequency impedance signatures. Biomass profiling of Cytodex-3 microcarriers seeded with adipose-derived mesenchymal stem cells (ADSCs) is first performed to sort well-seeded or confluent microcarriers for downstream culture or harvesting, respectively. Next, impedance-based isolation of microcarriers with osteogenic differentiated ADSCs is demonstrated, which is validated with a twofold increase of calcium content in sorted ADSCs. Impedance profiling of heterogenous ADSCs-encapsulated hydrogel (alginate) microparticles and 3D ADSC aggregate mixtures is also performed to sort particles with high biomass and cell viability to improve cell quality. Overall, the scalable microfluidic platform technology enables in-line sample processing from bioreactors directly and automated analysis of cell quality attributes to maximize cell yield and improve the control of cell quality in continuous cell-based manufacturing., (© 2024 Wiley‐VCH GmbH.)

Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc) for isolation of platelet-free plasma from blood for RNA and EV analysis. Unlike conventional inertial microfluidic devices widely used for cell sorting, a submicrometer size cutoff (500 nm) was achieved which completely removed all leukocytes, RBCs, platelets, and cellular debris based on differential lateral migration induced by Dean vortices. The single-step operation also reduced platelet-associated miRNAs (∼2-fold) compared to centrifugation. We clinically validated ExoArc for plasma miRNA profiling (39 samples) and identified a 7-miRNA panel that detects non-small cell lung cancer with ∼90% sensitivity. ExoArc was also coupled with size exclusion chromatography (SEC) to isolate EVs within 50 min with ∼10-fold higher yield than ultracentrifugation. As a proof-of-concept for EV-based transcriptomics analysis, we performed miRNA analysis in healthy and type 2 diabetes mellitus (T2DM) subjects ( n = 3 per group) by coupling ExoArc and ExoArc+SEC with quantitative polymerase chain reaction (RT-qPCR) assay. Among 293 miRNAs detected, plasmas and EVs showed distinct differentially expressed miRNAs in T2DM subjects. We further demonstrated automated in-line EV sorting from low volume culture media for continuous EV monitoring. Overall, the developed ExoArc offers a convenient centrifugation-free workflow to automate plasma and EV isolation for point-of-care diagnostics and quality control in EV manufacturing.

Insights into how biological systems respond to high- and low-dose acute environmental stressors are a fundamental aspect of exposome research. However, studying the impact of low-level environmental exposure in conventional in vitro settings is challenging. This study employed a three-dimensional (3D) biomimetic microfluidic lung-on-chip (μLOC) platform and RNA-sequencing to examine the effects of two model anthropogenic engineered nanoparticles (NPs): zinc oxide nanoparticles (Nano-ZnO) and copier center nanoparticles (Nano-CCP). The airway epithelium exposed to these NPs exhibited dose-dependent increases in cytotoxicity and barrier dysregulation (dominance of the external exposome). Interestingly, even nontoxic and low-level exposure (10 μg/mL) of the epithelium compartment to Nano-ZnO triggered chemotaxis of lung fibroblasts toward the epithelium. An increase in α smooth muscle actin (α-SMA) expression and contractile activity was also observed in these cells, indicating a bystander-like adaptive response (dominance of internal exposome). Further bioinformatics and network analysis showed that a low-dose Nano-ZnO significantly induced a robust transcriptomic response and upregulated several hub genes associated with the development of lung fibrosis. We propose that Nano-ZnO, even at a no observable effect level (NOEL) dose according to conventional standards, can function as a potent nanostressor to disrupt airway epithelium homeostasis. This leads to a cascade of profibrotic events in a cross-tissue compartment fashion. Our findings offer new insights into the early acute events of respiratory harm associated with environmental NPs exposure, paving the way for better exposomic understanding of this emerging class of anthropogenic nanopollutants.

Through extensive multisystem phenotyping, the central aim of Project PICMAN is to correlate metabolic flexibility to measures of cardiometabolic health, including myocardial diastolic dysfunction, coronary and cerebral atherosclerosis, body fat distribution and severity of non-alcoholic fatty liver disease. This cohort will form the basis of larger interventional trials targeting metabolic inflexibility in the prevention of cardiovascular disease. Participants aged 21-72 years with no prior manifest atherosclerotic cardiovascular disease (ASCVD) are being recruited from a preventive cardiology clinic and an existing cohort of non-alcoholic fatty liver disease (NAFLD) in an academic medical centre. A total of 120 patients will be recruited in the pilot phase of this study and followed up for 5 years. Those with 10-year ASCVD risk ≥ 5% as per the QRISK3 calculator are eligible. Those with established diabetes mellitus are excluded. Participants recruited undergo a detailed assessment of health behaviours and physical measurements. Participants also undergo a series of multimodality clinical phenotyping comprising cardiac tests, vascular assessments, metabolic tests, liver and neurovascular testing. Blood samples are also being collected and banked for plasma biomarkers, 'multi-omics analyses' and for generation of induced pluripotent stem cells (iPSC). Extensive evidence points to metabolic dysregulation as an early precursor of cardiovascular disease, particularly in Asia. We hypothesise that quantifiable metabolic inflexibility may be representative of an individual in his/her silent, but high-risk progression towards insulin resistance, diabetes and cardiovascular disease. The platform for interdisciplinary cardiovascular-metabolic-neurovascular diseases (PICMAN) is a pilot, prospective, multi-ethnic cohort study., (© 2023. The Author(s).)

The growing interest in regenerative medicine has opened new avenues for novel cell therapies using stem cells. Bone marrow aspirate (BMA) is an important source of stromal mesenchymal stem cells (MSCs). Conventional MSC harvesting from BMA relies on archaic centrifugation methods, often leading to poor yield due to osmotic stress, high centrifugation force, convoluted workflow, and long experimental time (∼2-3 hours). To address these issues, we have developed a scalable microfluidic technology based on deterministic lateral displacement (DLD) for MSC isolation. This passive, label-free cell sorting method capitalizes on the morphological differences between MSCs and blood cells (platelets and RBCs) for effective separation using an inverted L-shaped pillar array. To improve throughput, we developed a novel multi-chip DLD system that can process 2.5 mL of raw BMA in 20 ± 5 minutes, achieving a 2-fold increase in MSC recovery compared to centrifugation methods. Taken together, we envision that the developed DLD platform will enable fast and efficient isolation of MSCs from BMA for effective downstream cell therapy in clinical settings.

Neutrophils are the most abundant circulating white blood cells and one of their critical functions to eliminate pathogenic threats includes the release of extracellular DNA, also known as neutrophil extracellular traps (NETs), which is dysregulated in many diseases including cancer, type 2 diabetes mellitus and infectious diseases. Currently, conventional methods to quantify the NET formation (NETosis) rely on fluorescence antibody-based NET labelling or circulating NET-associated protein detection by ELISA, which are expensive, laborious, and time-consuming. In this work, we employed a novel "virtual staining" using deep convolutional neural networks (CNNs) to facilitate label-free quantification of NETs trapped in a micropillar array in a microfluidic device. Virtual staining is constructed to establish relations between morphological features in phase contrast images and fluorescence features in Sytox-green (DNA dye) images. We first investigated the effect of different learning rates on model training and optimized the learning rate to achieve the best model which can provide outputs close to Sytox green staining based on various reconstruction metrics ( e.g. , structural similarity (SSIM) and pixel-wise error (MAE, MSE)). The virtual staining of different NET concentrations was investigated which showed a linear correlation with fluorescent staining. As a proof of concept for clinical testing, the model was used to characterize purified neutrophils treated with NETosis inducers, including lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore (CaI), and successfully detected different NET profiles for different treatments. Collectively, these results demonstrated the potential of using deep learning for enhanced label-free image analysis of NETs for clinical research, drug discovery and point-of-care testing of diseases.

Urinary tract infection (UTI) diagnosis based on urine culture for bacteriuria analysis is time-consuming and often leads to wastage of hospital resources due to false-positive UTI cases. Direct cellular phenotyping (e.g., RBCs, neutrophils, epithelial cells) of urine samples remains a technical challenge as low cell concentrations, and urine characteristics (conductivities, pH, microbes) can affect the accuracy of cell measurements. In this work, we report a microfluidic inertial-impedance cytometry technique for label-free rapid (<5 min) neutrophil sorting and impedance profiling from urine directly. Based on size-based inertial focusing effects, neutrophils are isolated, concentrated, and resuspended in saline (buffer exchange) to improve consistency in impedance-based single-cell analysis. We first observed that both urine pH and the presence of bacteria can affect neutrophil high-frequency impedance measurements possibly due to changes in nucleus morphology as neutrophils undergo NETosis and phagocytosis, respectively. As a proof-of-concept for clinical testing, we report for the first time, rapid UTI testing based on multiparametric impedance profiling of putative neutrophils (electrical size, membrane properties, and distribution) in urine samples from non-UTI ( n = 20) and UTI patients ( n = 20). A significant increase in cell count was observed in UTI samples, and biophysical parameters were used to develop a UTI classifier with an area under the receiver operating characteristic curve of 0.84. Overall, the developed platform facilitates rapid culture-free urine screening which can be further developed to assess disease severity in UTI and other urologic diseases based on neutrophil electrical signatures.

Extracellular vesicles (EVs) can be produced from red blood cells (RBCs) on a large scale and used to deliver therapeutic payloads efficiently. However, not much is known about the native biological properties of RBCEVs. Here, we demonstrate that RBCEVs are primarily taken up by macrophages and monocytes. This uptake is an active process, mediated mainly by endocytosis. Incubation of CD14+ monocytes with RBCEVs induces their differentiation into macrophages with an Mheme-like phenotype, characterized by upregulation of heme oxygenase-1 (HO-1) and the ATP-binding cassette transporter ABCG1. Moreover, macrophages that take up RBCEVs exhibit a reduction in surface CD86 and decreased secretion of TNF-α under inflammatory stimulation. The upregulation of HO-1 is attributed to heme derived from haemoglobin in RBCEVs. Heme is released from internalized RBCEVs in late endosomes and lysosomes via the heme transporter, HRG1. Consequently, RBCEVs exhibit the ability to attenuate foam cell formation from oxidized low-density lipoproteins (oxLDL)-treated macrophages in vitro and reduce atherosclerotic lesions in ApoE knockout mice on a high-fat diet. In summary, our study reveals the uptake mechanism of RBCEVs and their delivery of heme to macrophages, suggesting the potential application of RBCEVs in the treatment of atherosclerosis., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the minimalist known angiosperm to overcome this challenge and provide a model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana , as well as high-quality genome information. Further, we developed the plant-on-chip culture system and demonstrate the application of advanced technologies such as single-nucleus RNA-sequencing, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can decipher the core regulatory mechanisms of plant morphogenesis., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Blood tests are considered as standard clinical procedures to screen for markers of diseases and health conditions. However, the complex cellular background (>99.9% RBCs) and biomolecular composition often pose significant technical challenges for accurate blood analysis. An emerging approach for point-of-care blood diagnostics is utilizing "label-free" microfluidic technologies that rely on intrinsic cell properties for blood fractionation and disease detection without any antibody binding. A growing body of clinical evidence has also reported that cellular dysfunction and their biophysical phenotypes are complementary to standard hematoanalyzer analysis (complete blood count) and can provide a more comprehensive health profiling. In this review, we will summarize recent advances in microfluidic label-free separation of different blood cell components including circulating tumor cells, leukocytes, platelets and nanoscale extracellular vesicles. Label-free single cell analysis of intrinsic cell morphology, spectrochemical properties, dielectric parameters and biophysical characteristics as novel blood-based biomarkers will also be presented. Next, we will highlight research efforts that combine label-free microfluidics with machine learning approaches to enhance detection sensitivity and specificity in clinical studies, as well as innovative microfluidic solutions which are capable of fully integrated and label-free blood cell sorting and analysis. Lastly, we will envisage the current challenges and future outlook of label-free microfluidics platforms for high throughput multi-dimensional blood cell analysis to identify non-traditional circulating biomarkers for clinical diagnostics.

Vascular stenosis caused by atherosclerosis instigates activation and aggregation of platelets, eventually resulting in thrombus formation. Although antiplatelet drugs are commonly used to inhibit platelet activation and aggregation, they unfortunately cannot prevent recurrent thrombotic events in patients with atherosclerosis. This is partially due to the limited understanding of the efficacy of antiplatelet drugs in the complex hemodynamic environment of vascular stenosis. Conventional methods for evaluating the efficacy of antiplatelet drugs under stenosis either fail to simulate the hemodynamic environment of vascular stenosis characterized by high shear stress and recirculatory flow or lack spatial resolution in their analytical techniques to statistically identify and characterize platelet aggregates. Here we propose and experimentally demonstrate a method comprising an in vitro 3D stenosis microfluidic chip and an optical time-stretch quantitative phase imaging system for studying the efficacy of antiplatelet drugs under stenosis. Our method simulates the atherogenic flow environment of vascular stenosis while enabling high-resolution and statistical analysis of platelet aggregates. Using our method, we distinguished the efficacy of three antiplatelet drugs, acetylsalicylic acid (ASA), cangrelor, and eptifibatide, for inhibiting platelet aggregation induced by stenosis. Specifically, ASA failed to inhibit stenosis-induced platelet aggregation, while eptifibatide and cangrelor showed high and moderate efficacy, respectively. Furthermore, we demonstrated that the drugs tested also differed in their efficacy for inhibiting platelet aggregation synergistically induced by stenosis and agonists ( e.g. , adenosine diphosphate, and collagen). Taken together, our method is an effective tool for investigating the efficacy of antiplatelet drugs under vascular stenosis, which could assist the development of optimal pharmacologic strategies for patients with atherosclerosis.

Non-alcoholic steatohepatitis (NASH) is a global disease with no effective medication. The fibroblast growth factor 21 (FGF21) can reverse this liver dysfunction, but requires targeted delivery to the liver, which can be achieved via oral administration. Therefore, we fused FGF21 to transferrin (Tf) via a furin cleavage site (F), to promote uptake from the intestine into the portal vein, yielding FGF21-F-Tf, and established its production in both seeds and leaves of commercial Nicotiana tabacum cultivars, compared their expression profile and tested the bioavailability and bioactivity in feeding studies. Since biopharmaceuticals need to be produced in a contained environment, e.g., greenhouses in case of plants, the seed production was increased in this setting from 239 to 380 g m -2 a -1 seed mass with costs of 1.64 € g -1 by side branch induction, whereas leaves yielded 8,193 g m -2 a -1 leave mass at 0.19 € g -1 . FGF21-F-Tf expression in transgenic seeds and leaves yielded 6.7 and 5.6 mg kg -1 intact fusion protein, but also 4.5 and 2.3 mg kg -1 additional Tf degradation products. Removing the furin site and introducing the liver-targeting peptide PLUS doubled accumulation of intact FGF21-transferrin fusion protein when transiently expressed in Nicotiana benthamiana from 0.8 to 1.6 mg kg -1 , whereas truncation of transferrin (nTf338) and reversing the order of FGF21 and nTf338 increased the accumulation to 2.1 mg kg -1 and decreased the degradation products to 7% for nTf338-FGF21-PLUS. Application of partially purified nTf338-FGF21-PLUS to FGF21 -/- mice by oral gavage proved its transfer from the intestine into the blood circulation and acutely affected hepatic mRNA expression. Hence, the medication of NASH via oral delivery of nTf338-FGF21-PLUS containing plants seems possible., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hou, Bishop, Huckauf, Broer, Klaus, Nausch and Buyel.)

The intrinsic biophysical states of neutrophils are associated with immune dysfunctions in diseases. While advanced image-based biophysical flow cytometers can probe cell deformability at high throughput, it is nontrivial to couple different sensing modalities (e.g., electrical) to measure other critical cell attributes including cell viability and membrane integrity. Herein, an "optics-free" impedance-deformability cytometer for multiparametric single cell mechanophenotyping is reported. The microfluidic platform integrates hydrodynamic cell pinching, and multifrequency impedance quantification of cell size, deformability, and membrane impedance (indicative of cell viability and activation). A newly-defined "electrical deformability index" is validated by numerical simulations, and shows strong correlations with the optical cell deformability index of HL-60 experimentally. Human neutrophils treated with various biochemical stimul are further profiled, and distinct differences in multimodal impedance signatures and UMAP analysis are observed. Overall, the integrated cytometer enables label-free cell profiling at throughput of >1000 cells min -1 without any antibodies labeling to facilitate clinical diagnostics., (© 2022 Wiley-VCH GmbH.)

Background: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis., Results: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples., Conclusions: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin., (© 2022. The Author(s).)

Extracellular vesicles (EVs) are recognized as next generation diagnostic biomarkers due to their disease-specific biomolecular cargoes and importance in cell-cell communications. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of nanoscale EVs (≈50-200 nm) from endogenous proteins in biological samples. Herein, a unique microfluidic platform is reported for EV-protein fractionation based on the principle of size exclusion chromatography (SEC). Using a novel rapid (≈20 min) replica molding technique, a fritless microfluidic SEC device (μSEC) is fabricated using thiol-ene polymer (UV glue NOA81, Young's modulus ≈1 GPa) for high pressure (up to 6 bar) sample processing. Controlled on-chip nanoliter sample plug injection (600 nL) using a modified T-junction injector is first demonstrated with rapid flow switching response time (<1.5 s). Device performance is validated using fluorescent nanoparticles (50 nm), albumin, and breast cancer cells (MCF-7)-derived EVs. As a proof-of-concept for clinical applications, EVs are directly isolated from undiluted human platelet-poor plasma using μSEC and show distinct elution profiles between EVs and proteins based on nanoparticle particle analysis (NTA), Western blot and flow cytometry analysis. Overall, the optically transparent μSEC can be readily automated and integrated with EV detection assays for EVs manufacturing and clinical diagnostics., (© 2022 Wiley-VCH GmbH.)

Incorporation of extracellular matrix (ECM) and hydrogel in microfluidic 3D cell culture platforms is important to create a physiological microenvironment for cell morphogenesis and to establish 3D co-culture models by hydrogel compartmentalization. Here, we describe a simple and scalable ECM patterning method for microfluidic cell cultures by achieving hydrogel confinement due to the geometrical expansion of channel heights (stepped height features) and capillary burst valve (CBV) effects. We first demonstrate a sequential "pillar-free" hydrogel patterning to form adjacent hydrogel lanes in enclosed microfluidic devices, which can be further multiplexed with one to two stepped height features. Next, we developed a novel "spheroid-in-gel" culture device that integrates (1) an on-chip hanging drop spheroid culture and (2) a single "press-on" hydrogel confinement step for rapid ECM patterning in an open-channel microarray format. The initial formation of breast cancer (MCF-7) spheroids was achieved by hanging a drop culture on a patterned polydimethylsiloxane (PDMS) substrate. Single spheroids were then directly encapsulated on-chip in individual hydrogel islands at the same positions, thus, eliminating any manual spheroid handling and transferring steps. As a proof-of-concept to perform a spheroid co-culture, endothelial cell layer (HUVEC) was formed surrounding the spheroid-containing ECM region for drug testing studies. Overall, this developed stepped height-based hydrogel patterning method is simple to use in either enclosed microchannels or open surfaces and can be readily adapted for in-gel cultures of larger 3D cellular spheroids or microtissues.

Butterfly-like molecules of oxacalix[2]arene[2]pyrazine (OAP) are reported, which exhibit the typical characteristics of aggregation-induced emission (AIE) via the restriction of intramolecular vibration (RIV) mechanism. Unlike any of the reported RIV-type AIE molecules, the synthetic procedures of which are complicated and have associated high costs, OAP AIEgens can be synthesized in a facile manner by a one-step catalyst-free reaction using commercially available materials. Notably, OAP AIEgens are ideal ligands for constructing metal-organic frameworks (MOFs) due to their built-in pyrazine coordination sites. OAP-based MOFs exhibit multiple potential applications in reversible gas response, encrypted information storage, and construction of white light-emitting devices. This work builds on RIV-type AIEgens, offers additional selections of bridging ligands for constructing luminescent MOFs and provides a visualized prototype to understand the effect of the RIV process on the luminescence properties of MOFs., (© 2021 Wiley-VCH GmbH.)

Single-cell metabolite measurement remains highly challenging due to difficulties related to single cell isolation, metabolite detection, and identification of low levels of metabolites. Here, as a first step of the technological development, we propose a novel strategy integrating spiral inertial microfluidics and ion mobility mass spectrometry (IM-MS) for single-cell metabolite detection and identification. Cells in methanol suspension are inertially focused into a single stream in the spiral microchannel. This stream of separated cells is delivered to the nanoelectrospray needle to be lysed and ionized and subsequently analyzed in real time by IM-MS. This analytical system enables six to eight single-cell metabolic fingerprints to be collected per minute, including gas-phase collisional cross section (CCS) measurements as an additional molecular descriptor, giving increased confidence in metabolite identification. As a proof of concept, the metabolic profiles of three types of cancer cells (U2OS, HepG2, and HepG2.215) were successfully screened, and 19 distinct lipids species were identified with CCS value filtering. Furthermore, principal component analysis (PCA) showed differentiation of the three cancer cell lines, mainly due to cellular surface phospholipids. Taken together, our technology platform offers a simple and efficient method for single-cell lipid profiling, with additional ion mobility separation of lipids significantly improving the confidence toward identification of metabolites.

Elevated serum concentrations of leucine-rich α-2-glycoprotein (LRG1) have been reported in patients with inflammatory, autoimmune, and cardiovascular diseases. This study aims to investigate the role of LRG1 in endothelial activation. LRG1 in endothelial cells (ECs) of arteries and serum of patients with critical limb ischemia (CLI) was assessed by immunohistochemistry and ELISA, respectively. LRG1 expression in sheared and tumor necrosis factor-α (TNF-α)-treated ECs was analyzed. The mechanistic role of LRG1 in endothelial activation was studied in vitro . Plasma of 37-week-old Lrg1 -/- mice was used to investigate causality between LRG1 and tumor necrosis factor receptor 1 (TNFR1) shedding. LRG1 was highly expressed in ECs of stenotic but not normal arteries. LRG1 concentrations in serum of patients with CLI were elevated compared to healthy controls. LRG1 expression was shear dependent. It could be induced by TNF-α, and the induction of its expression was mediated by NF-κB activation. LRG1 inhibited TNF-α-induced activation of NF-κB signaling, expression of VCAM-1 and ICAM-1, and monocyte capture, firm adhesion, and transendothelial migration. Mechanistically, LRG1 exerted its function by causing the shedding of TNFR1 via the ALK5-SMAD2 pathway and the subsequent activation of ADAM10. Consistent with this mechanism, LRG1 and sTNFR1 concentrations were correlated in the serum of CLI patients. Causality between LRG1 and TNFR1 shedding was established by showing that Lrg1 -/- mice had lower plasma sTNFR1 concentrations than wild type mice. Our results demonstrate a novel role for LRG1 in endothelial activation and its potential therapeutic role in inflammatory diseases should be investigated further., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pang, Ghim, Liu, Tay, Fhu, Chia, Qiu, Sarathchandra, Chester, Yacoub, Wilkinson, Weston, Warboys, Hou, Weinberg and Wang.)

Sialyl-Lewis x (sLe x , CD15s) is a tetra-saccharide on the surface of leukocytes required for E-selectin-mediated rolling, a prerequisite for leukocytes to migrate out of the blood vessels. Here we show using flow cytometry that sLe x expression on basophils and mast cell progenitors depends on fucosyltransferase 6 (FUT6). Using genetic association data analysis and qPCR, the cell type-specific defect was associated with single nucleotide polymorphisms (SNPs) in the FUT6 gene region (tagged by rs17855739 and rs778798), affecting coding sequence and/or expression level of the mRNA. Heterozygous individuals with one functional FUT6 gene harbor a mixed population of sLe x+ and sLe x- basophils, a phenomenon caused by random monoallelic expression (RME). Microfluidic assay demonstrated FUT6-deficient basophils rolling on E-selectin is severely impaired. FUT6 null alleles carriers exhibit elevated blood basophil counts and a reduced itch sensitivity against insect bites. FUT6-deficiency thus dampens the basophil-mediated allergic response in the periphery, evident also in lower IgE titers and reduced eosinophil counts.

Extracellular vesicles (EVs) are key mediators of communication among cells, and clinical utilities of EVs-based biomarkers remain limited due to difficulties in isolating EVs from whole blood reliably. We report a novel inertial-based microfluidic platform for direct isolation of nanoscale EVs (exosomes, 50 to 200 nm) and medium-sized EVs (microvesicles, 200 nm to 1 μm) from blood with high efficiency (three-fold increase in EV yield compared to ultracentrifugation). In a pilot clinical study of healthy (n = 5) and type 2 diabetes mellitus (T2DM, n = 9) subjects, we detected higher EV levels in T2DM patients (P < 0.05), and identified a subset of "high-risk" T2DM subjects with abnormally high (∼10-fold to 50-fold) amounts of platelet (CD41a+) or leukocyte-derived (CD45+) EVs. Our in vitro endothelial cell assay further revealed that EVs from "high-risk" T2DM subjects induced significantly higher vascular inflammation (ICAM-1 expression) (P < 0.05) as compared to healthy and non-"high-risk" T2DM subjects, reflecting a pro-inflammatory phenotype. Overall, the EV isolation tool is scalable, and requires less manual labour, cost and processing time. This enables further development of EV-based diagnostics, whereby a combined immunological and functional phenotyping strategy can potentially be used for rapid vascular risk stratification in T2DM.

Mechanistic understanding of atherosclerosis is largely hampered by the lack of a suitable in vitro human arterial model that recapitulates the arterial wall structure, and the interplay between different cell types and the surrounding extracellular matrix (ECM). This work introduces a novel microfluidic endothelial cell (EC)-smooth muscle cell (SMC) 3D co-culture platform that replicates the structural and biological aspects of the human arterial wall for modeling early atherosclerosis. Using a modified surface tension-based ECM patterning method, we established a well-defined intima-media-like structure, and identified an ECM composition (collagen I and Matrigel mixture) that retains the SMCs in a quiescent and aligned state, characteristic of a healthy artery. Endothelial stimulation with cytokines (IL-1β and TNFα) and oxidized low-density lipoprotein (oxLDL) was performed on-chip to study various early atherogenic events including endothelial inflammation (ICAM-1 expression), EC/SMC oxLDL uptake, SMC migration, and monocyte-EC adhesion. As a proof-of-concept for drug screening applications, we demonstrated the atheroprotective effects of vitamin D (1,25(OH)2D3) and metformin in mitigating cytokine-induced monocyte-EC adhesion and SMC migration. Overall, the developed arterial wall model facilitates quantitative and multi-factorial studies of EC and SMC phenotype in an atherogenic environment, and can be readily used as a platform technology to reconstitute multi-layered ECM tissue biointerfaces.

3D cellular spheroids/microcarriers (100 µm-1 mm) are widely used in biomanufacturing, and non-invasive biosensors are useful to monitor cell quality in bioprocesses. In this work, a novel microfluidic approach for label-free and continuous-flow monitoring of single spheroid/microcarrier (hydrogel and Cytodex) based on electrical impedance spectroscopy using co-planar Field's metal electrodes is reported. Through numerical simulation and experimental validation, two unique impedance signatures (|Z LF | (60 kHz), |Z HF | (1 MHz)) which are optimal for spheroid growth and viability monitoring are identified. Using a closed-loop recirculation system, it is demonstrated that |Z LF | increases with breast cancer (MCF-7) spheroid biomass, while higher opacity (impedance ratio |Z HF |/|Z LF |) indicates cell death due to compromised cell membrane. Anti-cancer drug (paclitaxel)-treated spheroids also exhibit lower |Z LF | with increased cell dissociation. Interestingly, impedance characterization of adipose-derived mesenchymal stem cell differentiation on Cytodex microcarriers reveals that adipogenic cells (higher intracellular lipid content) exhibit higher impedance than osteogenic cells (more conductive due to calcium ions) for both microcarriers and single cell level. Taken together, the developed platform offers great versatility for multi-parametric analysis of spheroids/microcarriers at high throughput (≈1 particle/s), and can be readily integrated into bioreactors for long-term and remote monitoring of biomass and cell quality., (© 2021 Wiley-VCH GmbH.)

Objective: This study aims to investigate whether liraglutide can affect proliferation, osteogenic differentiation and serum deprivation-induced apoptosis of preosteoblast cell line MC3T3-E1 through the Notch, Wnt/β-catenin, and Hedgehog (Hh) signaling pathways., Materials and Methods: MC3T3-E1 cells were exposed to different treatments (via Notch inhibitor DAPT, an Hh inhibitor cyclopamine, or serum deprivation) or transfections of different siRNAs (targeting glucagon-like peptide-1 receptor (GLP-1R), β-catenin, or Gli1) in the presence or absence of 100 nM liraglutide. Cell proliferation, mRNA levels of osteogenic differentiation-related genes, mRNA and protein levels of the Notch and Hh signaling pathway proteins, and apoptosis-related proteins were assessed., Results: Liraglutide significantly increased proliferation of MC3T3-E1 cells, expression levels of the Notch and Hh signaling pathway proteins and β-catenin, and mRNA levels of osteogenic differentiation-related genes and TCF7L2. Moreover, liraglutide promoted a translocation of β-catenin, increased a ratio of Bcl-2/Bax proteins, reduced serum deprivation-induced apoptosis of MC3T3-E1 cells, and a ratio of caspase-3/procaspase-3. However, a cotreatment with liraglutide and DAPT reversed the alterations. A cyclopamine treatment and knockdowns of GLP-1R, Gli1, and β-catenin significantly reduced the expression of Notch proteins. Furthermore, the knockdown of GLP-1R, β-catenin, or Gli1 significantly increased apoptosis, which could be inhibited by liraglutide., Conclusions: In summary, liraglutide can promote proliferation and differentiation of MC3T3-E1 cells, and inhibit their serum deprivation-induced apoptosis by activating both the Notch and Hh signaling pathways involving β-catenin and Gli1. These results provide a therapeutic foundation that patients with diabetes and osteoporosis may be cured with treatments of liraglutide.

Delayed wound healing is commonly associated with diabetes. It may lead to amputation and death if not treated in a timely fashion. Limited treatments are available partially due to the poor understanding of the complex disease pathophysiology. Here, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in normal and diabetic wound healing. First, our data showed that LRG1 was significantly increased at the inflammation stage of murine wound healing, and bone marrow-derived cells served as a major source of LRG1. LRG1 deletion causes impaired immune cell infiltration, reepithelialization, and angiogenesis. As a consequence, there is a significant delay in wound closure. On the other hand, LRG1 was markedly induced in diabetic wounds in both humans and mice. LRG1-deficient mice were resistant to diabetes-induced delay in wound repair. We further demonstrated that this could be explained by the mitigation of increased neutrophil extracellular traps (NETs) in diabetic wounds. Mechanistically, LRG1 mediates NETosis in an Akt-dependent manner through TGFβ type I receptor kinase ALK5. Taken together, our studies demonstrated that LRG1 derived from bone marrow cells is required for normal wound healing, revealing a physiological role for this glycoprotein, but that excess LRG1 expression in diabetes is pathogenic and contributes to chronic wound formation., (© 2020 by the American Diabetes Association.)

Blood vessel narrowing and arterial occlusion are pathological hallmarks of atherosclerosis, which involves a complex interplay of perturbed hemodynamics, endothelial dysfunction and inflammatory cascade. Herein, we report a novel circular microfluidic stenosis model that recapitulates atherogenic flow-mediated endothelial dysfunction and blood-endothelial cell (EC) interactions in vitro. 2D and 3D stenosis microchannels with different constriction geometries were fabricated using 3D printing to study flow disturbances under varying severity of occlusion and wall shear stresses (100 to 2000 dynecm -2 ). Experimental and fluid simulation results confirmed the presence of pathological shear stresses in the stenosis region, and recirculation flow post stenosis. The resultant pathological flow profile induced pro-inflammatory and pro-thrombotic EC state as demonstrated by orthogonal EC alignment, enhanced platelet adhesion at the stenosis, and aberrant leukocyte-EC interactions post stenosis. Clinical utility of the vascular model was further investigated by testing anti-thrombotic and immunomodulatory efficacy of aspirin and metformin, respectively. Overall, the platform enables multi-factorial analysis of critical atherogenic events including endothelial dysfunction, platelets and leukocyte adhesion, and can be further developed into a liquid biopsy tool for cardiovascular risk stratification.

Exogenous sources of amino acids are essential nutrients to fuel cancer growth. Here, the increased demand for amino acid displayed by cancer cells is unconventionally exploited as a design principle to replete cancer cells with apoptosis inducing nanoscopic porous amino acid mimics (Nano-PAAM). A small library consisting of nine essential amino acids nanoconjugates (30 nm) are synthesized, and the in vitro anticancer activity is evaluated. Among the Nano-PAAMs, l-phenylalanine functionalized Nano-PAAM (Nano-pPAAM) has emerged as a novel nanotherapeutics with excellent intrinsic anticancer and cancer-selective properties. The therapeutic efficacy of Nano-pPAAM against a panel of human breast, gastric, and skin cancer cells could be ascribed to the specific targeting of the overexpressed human large neutral amino acid transporter SLC7A5 (LAT-1) in cancer cells, and its intracellular reactive oxygen species (ROS) inducing properties of the nanoporous core. At the mechanistic level, it is revealed that Nano-pPAAM could activate both the extrinsic and intrinsic apoptosis pathways to exert a potent "double-whammy" anticancer effect. The potential clinical utility of Nano-pPAAM is further investigated using an MDA-MB-231 xenograft in NOD scid gamma mice, where an overall suppression of tumor growth by 60% is achieved without the aid of any drugs or application of external stimuli., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vitamin D deficiency is a major public health problem worldwide, linked to several chronic diseases including cardiovascular diseases. While immunomodulatory effects of vitamin D on monocytes have been reported in cardiovascular and metabolic diseases, there is limited understanding on monocyte phenotype in healthy individuals with suboptimal vitamin D levels and without any clinical diseases. In this work, we performed label-free, microfluidic isolation of monocytes, and characterized their functional phenotype using flow cytometry and in vitro vascular models in healthy subjects with (n = 7) and without vitamin D deficiency (n = 16). Vitamin D deficient (VitD-Def) subjects (25(OH)D 3 level < 26 ng/mL) expressed significant downregulation of vitamin D receptor (VDR) on monocytes as compared to controls (P < .0001), and VDR expression was well-associated with serum 25(OH)D 3 levels. Increased monocyte-platelet aggregates (MPA), a marker for platelet activation, were also observed in VitD-Def subjects (P < .05) which suggests a pro-inflammatory monocyte phenotype. Monocyte adhesion to endothelial cells, an early-stage atherosclerosis event, was also higher in VitD-Def individuals, and inversely correlated to serum 25(OH)D 3 level (P < .05). Taken together, these results indicate the pro-inflammatory state and atherogenic potential of monocytes in VitD-Def healthy subjects, and propound the use of vitamin D supplementation as a prospective immunomodulatory and anti-inflammatory therapy in atherosclerosis., (© 2020 Federation of American Societies for Experimental Biology.)

Glaucoma is characterized by retinal ganglion cell (RGC) degeneration and is the second leading cause of blindness worldwide. However, current treatments such as eye drop or surgery have limitations and do not target the loss of RGC. Regenerative therapy using embryonic stem cells (ESCs) holds a promising option, but ethical concern hinders clinical applications on human subjects. In this study, we employed spermatogonial stem cells (SSCs) as an alternative source of ESCs for cell-based regenerative therapy in mouse glaucoma model. We generated functional RGCs from SSCs with a two-step protocol without applying viral transfection or chemical induction. SSCs were first dedifferentiated to embryonic stem-like cells (SSC-ESCs) that resemble ESCs in morphology, gene expression signatures, and stem cell properties. The SSC-ESCs then differentiated toward retinal lineages. We showed SSC-ESC-derived retinal cells expressed RGC-specific marker Brn3b and functioned as bona fide RGCs. To allow in vivo RGC tracing, Brn3b-EGFP reporter SSC-ESCs were generated and the derived RGCs were subsequently transplanted into the retina of glaucoma mouse models by intravitreal injection. We demonstrated that the transplanted RGCs could survive in host retina for at least 10 days after transplantation. SSC-ESC-derived RGCs can thus potentially be a novel alternative to replace the damaged RGCs in glaucomatous retina.

Changing the pH value of a reaction system can result in polymers with very different compositions and architectures. Two new coordination polymers based on 1,1'-[1,4-phenylenebis(methylene)]bis(3,5-dicarboxylatopyridinium) (L 2- ), namely catena-poly[[[tetraaquacadmium(II)]-μ 2 -1,1'-[1,4-phenylenebis(methylene)]bis(3,5-dicarboxylatopyridinium)] 1.66-hydrate], {[Cd(C 22 H 14 N 2 O 8 )(H 2 O) 4 ]·1.66H 2 O} n , (I), and poly[{μ 6 -1,1'-[1,4-phenylenebis(methylene)]bis(3,5-dicarboxylatopyridinium)}cadmium(II)], [Cd(C 22 H 14 N 2 O 8 )] n , (II), have been prepared in the presence of NaOH or HNO 3 and structurally characterized by single-crystal X-ray diffraction. In polymer (I), each Cd II ion is coordinated by two halves of independent L 2- ligands, forming a one-dimensional chain structure. In the crystal, these chains are further connected through O-H...O hydrogen bonds, leading to a three-dimensional hydrogen-bonded network. In polymer (II), each hexadentate L 2- ligand coordinates to six Cd II ions, resulting in a three-dimensional network structure, in which all of the Cd II ions and L 2- ligands are equivalent, respectively. The IR spectra, thermogravimetric analyses and fluorescence properties of both reported compounds were investigated.

Aminobenzoic acid derivatives are widely used in the preparation of new coordination polymers since they contain O-atom donors, as well as N-atom donors, and have a rich variety of coordination modes which can lead to polymers with intriguing structures and interesting properties. Two new coordination polymers incorporating 1-aminobenzene-3,4,5-tricarboxylic acid (H 3 abtc), namely, poly[(μ 3 -1-amino-5-carboxybenzene-3,4-dicarboxylato)diaquacadmium(II)], [Cd(C 9 H 5 NO 6 )(H 2 O) 2 ] n , (I), and poly[[bis(μ 5 -1-aminobenzene-3,4,5-tricarboxylato)triaquatrizinc(II)] dihydrate], {[Zn 3 (C 9 H 4 NO 6 ) 2 (H 2 O) 3 ]·2H 2 O} n , (II), have been prepared and structurally characterized by single-crystal X-ray diffraction. In polymer (I), each tridentate 1-amino-5-carboxybenzene-3,4-dicarboxylate (Habtc 2- ) ligand coordinates to three Cd II ions to form a two-dimensional network structure, in which all of the Cd II ions and Habtc 2- ligands are equivalent, respectively. Polymer (II) also exhibits a two-dimensional network structure, in which three crystallographically independent Zn II ions are bridged by two crystallographically independent pentadentate 1-aminobenzene-3,4,5-tricarboxylate (abtc 3- ) ligands. This indicates that changing the metal ion can influence the coordination mode of the H 3 abtc-derived ligand and further influence the detailed architecture of the polymer. Moreover, the IR spectra, thermogravimetric analyses and fluorescence properties were investigated.

Anastomotic fibrosis is highly likely to lead to reoperation in Crohn's disease (CD) patients. Triptolide (TPL) is considered to have anti‑inflammatory and antifibrotic effects in a variety of autoimmune diseases, including CD. The present study aimed to investigate the effects of TPL on fibroblasts from strictured ileocolonic anastomosis of patients with CD and its underlying mechanism. Primary fibroblasts were obtained from strictured anastomosis tissue (SAT) samples and matched anastomosis‑adjacent normal tissue (NT) samples which were collected from 10 CD patients who underwent reoperation because of anastomotic stricture. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to measure miR‑16‑1 and heat shock protein 70 (HSP70) levels. Western blotting was conducted to determine expression of HSP70, collagen I (Col‑I), collagen III (Col‑III) and α‑smooth muscle actin (α‑SMA) proteins. Agomir‑16‑1 and antagomir‑16‑1 were used to up and downregulate the expression of miR‑16‑1, respectively. Small interfering RNA (siRNA) was employed to inhibit the expression of HSP70. A wound healing assay was performed to measure the migration of fibroblasts. Cell proliferation was evaluated by MTT and 5‑bromo‑2‑deoxyrudidine assays. Cell apoptosis was determined by caspase‑3 activity and TUNEL assays. The results demonstrated that the levels of Col‑I, Col‑III and α‑SMA were all significantly upregulated in SAT compared with NT. miR‑16‑1 levels in the SAT group were significantly compared with the NT group; conversely, the expression levels of HSP70 mRNA and protein in the SAT group were significantly lower compared with the NT group. Next, fibroblasts were treated with TPL to examine its effect on the miR‑16‑1/HSP70 pathway. The results demonstrated that the elevated expression of miR‑16‑1 in the SAT group was effectively inhibited by TPL treatment. Compared with the NT group, both the mRNA and protein levels of HSP70 were significantly downregulated in the SAT group cells, while TPL exhibited a strong promoting effect on HSP70 synthesis. Furthermore, upregulation of miR‑16‑1 reversed the effect of TPL on the miR‑16‑1/HSP70 pathway in fibroblasts from SAT. Overexpression of miR‑16‑1 significantly reversed the inhibitory effects of TPL treatment on migration, proliferation and extracellular matrix (ECM)‑associated protein expression of fibroblasts from SAT. Finally, downregulation of miR‑16‑1 caused similar effects to the fibroblasts as the TPL treatment; however, the inhibitory effects on cell biological functions induced by antagomir‑16‑1 were all significantly reversed by HSP70 silencing. The present findings indicated that TPL may be a potential therapeutic option for postoperative anastomosis fibrosis of patients with CD. The miR‑16‑1/HSP70 pathway had a substantial role in the inhibitory effects of TPL on migration, proliferation and ECM synthesis rate of fibroblasts from strictured anastomosis tissues.

Circulating leukocytes are indispensable components of the immune system, and rapid analysis of their native state or functionalities can help to unravel their pathophysiological roles and identify novel prognostic biomarkers in health and diseases. Herein we report a novel high throughput "sample-in-answer-out" integrated platform for continuous leukocyte sorting and single-cell electrical profiling in a label-free manner. The multi-staged platform enables isolation of neutrophils and monocytes from diluted or lysed blood samples directly within minutes based on Dean flow fractionation (DFF) (stage 1). Next DFF-purified leukocytes are inertially focused in serpentine channels into a single stream (stage 2) prior to impedance detection (stage 3). As a proof-of-concept for neutrophil functional characterization towards diabetes testing, we characterized the formation of neutrophil extracellular traps (NETosis) of healthy and glucose-treated neutrophils and observed significant changes in dielectric properties (size and opacity) between both groups. Interestingly, the NETosis profiles induced by calcium ionophore (CaI) and phorbol 12-myristate 13-acetate (PMA) were also electrically different, which could be attributed to the differential rates of cell enlargement and attenuated membrane permeability. Taken together, these results clearly demonstrated the potential of the developed platform for rapid (∼mins) and label-free leukocyte profiling and the use of impedance signatures as novel functional biomarkers for point-of-care testing in diabetes.

Monocytes and platelets play key roles in atherosclerosis and thrombosis, and circulating monocyte-platelet aggregates (MPA) in blood have been widely proposed as surrogate biomarkers for cardiovascular risk stratification and monitoring antiplatelet therapies. However, conventional MPA characterization is based on whole-blood fixation and flow cytometry analysis which adversely affect cell viability and detection accuracy due to significant leukocyte and platelet contaminations. Herein, we introduce a rapid and label-free microfluidic approach for complete size-based fractionation of peripheral blood mononuclear cells (PBMCs) into monocytes, lymphocytes, and platelets. The developed biochip enables gentle sorting of intact MPA in the enriched monocytes with efficient depletion of lymphocytes and platelets for accurate MPA quantification. We first compared the developed microfluidic technology (dean flow fractionation, DFF) with standard magnetic negative isolation (MACS) and observed that DFF-sorted monocytes had similar viability, purity, and key immune functions (phagocytosis, macrophage differentiation) as MACS-sorted monocytes. As proof of concept for diabetes testing, we isolated and characterized monocytes/MPA from a cohort of healthy ( n = 5) and type 2 diabetes mellitus (T2DM) subjects ( n = 8) in PBMCs and DFF-sorted monocytes. High-speed imaging, immunofluorescence, and flow cytometry analysis clearly indicated higher levels of MPA in T2DM patients ( P < 0.05) with enhanced MPA detection sensitivity in DFF-sorted monocytes ( P < 0.05). Taken together, the developed DFF technology greatly facilitates high-throughput (∼130 μL min -1 ) label-free isolation of monocyte/MPA from PBMCs and can be further developed into a clinical tool for point-of-care cardiovascular risk stratification in metabolic disorders including T2DM.

Circulating leukocytes comprise of approximately 1% of all blood cells and efficient enrichment of these cells from whole blood is critical for understanding cellular heterogeneity and biological significance in health and diseases. In this work, we report a novel microfluidic strategy for rapid (< 1 h) label-free leukocyte sorting and impedance-based profiling to determine cell activation in type 2 diabetes mellitus (T2DM) using whole blood. Leukocytes were first size-fractionated into different subtypes (neutrophils, monocytes, lymphocytes) using an inertial spiral sorter prior to single-cell impedance measurement in a microfluidic device with coplanar electrode design. Significant changes in membrane dielectric properties (size and opacity) were detected between healthy and activated leukocytes (TNF-α/LPS stimulated), during monocyte differentiation and among different monocyte subsets (classical, intermediate, non-classical). As proof-of-concept for diabetes testing, neutrophil/monocyte dielectric properties in T2DM subjects (n = 8) were quantified which were associated with cardiovascular risk factors including lipid levels, C-reactive protein (CRP) and vascular functions (LnRHI) (P < 0.05) were observed. Overall, these results clearly showed that T2DM subjects have pro-inflammatory leukocyte phenotypes and suggest leukocyte impedance signature as a novel surrogate biomarker for inflammation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The molecular structures of tetra-aqua-[ N , N '-bis-(pyridin-4-yl)pyridine-2,6-dicar-boxamide]-sulfatomanganese(II) dihydrate, [Mn(SO 4 )(C 17 H 13 N 5 O 2 )(H 2 O) 4 ]·2H 2 O or [Mn(H 2 L 1 )(SO 4 )(H 2 O) 4 ]·2H 2 O, (I), and tetra-aqua-bis[ N , N '-bis-(pyridin-4-yl)pyridine-2,6-dicar-boxamide]cadmium(II) sulfate tetra-hydrate, [Cd(C 17 H 13 N 5 O 2 ) 2 (H 2 O) 4 ]SO 4 ·4H 2 O or [Cd(H 2 O) 4 (H 2 L 1 ) 2 ]·SO 4 ·4H 2 O, (II), both contain a central metal atom in a distorted octa-hedral geometry coordinated equatorially by four oxygen atoms from water mol-ecules. In (I), the axial positions are occupied by a nitro-gen atom from H 2 L 1 and an oxygen atom from the sulfate anion, whereas in (II), the axial positions contain two nitro-gen atoms from two different H 2 L 1 ligands and the sulfate anion acts as the charge-balancing ion. π-π stacking between pyridine rings and a network of hydrogen bonds involving the water molecules and the sulfate anions play a crucial role in the mol-ecular self-assembly of the two structures.

Purpose: To investigate the temporal relationship among progressive macular ganglion cell inner plexiform layer (GCIPL) thinning, progressive parapapillary retinal nerve fiber layer (RNFL) thinning, and visual field (VF) progression in patients with primary open-angle glaucoma (POAG)., Design: Prospective study., Participants: One hundred thirty-six POAG patients (231 eyes) followed up for ≥5 years., Methods: OCT imaging of the macular GCIPL and parapapillary RNFL and perimetry were performed at ∼ 4-month intervals. Progressive GCIPL and RNFL thinning were determined by Guided Progression Analysis (GPA) of serial GCIPL and RNFL thickness maps. The specificities of GPA were calculated from the proportions of eyes with progressive GCIPL or RNFL thinning in 67 eyes of 36 healthy individuals followed up for ≥5 years. Visual field progression (likely or possible) was determined by the Early Manifest Glaucoma Trial criteria., Main Outcome Measures: Hazard ratios for VF progression, progressive RNFL thinning, and progressive GCIPL thinning, as determined by time-varying Cox models., Results: GPA detected 57 eyes (24.7%) with progressive GCIPL thinning and 66 eyes (28.6%) with progressive RNFL thinning at a specificity of 95.5% and 91.0%, respectively. Thirty-five eyes (15.2%) demonstrated progressive RNFL and GCIPL thinning, whereas 53 eyes (22.9%) demonstrated progressive RNFL or GCIPL thinning. Eyes with progressive GCIPL thinning had a higher risk for progressive RNFL thinning (HR, 5.27; 95% confidence interval [CI], 2.89-9.62), whereas eyes with progressive RNFL thinning were also at a higher risk for progressive GCIPL thinning (HR, 2.99; 95% CI, 1.48-6.02), after adjusting for baseline covariates. The HRs for likely and possible VF progression were 3.48 (95% CI, 1.51-8.01) and 2.74 (95% CI, 1.26-5.98), respectively, on detection of progressive GCIPL thinning and 3.66 (95% CI, 1.68-7.97) and 2.54 (95% CI, 1.23-5.21), respectively, on detection of progressive RNFL thinning after adjusting for baseline covariates. Eyes with VF progression were not at risk of progressive RNFL or GCIPL thinning (P ≥ 0.493)., Conclusions: Progressive macular GCIPL thinning and progressive parapapillary RNFL thinning are mutually predictive. Because progressive RNFL thinning and progressive GCIPL thinning are both indicative of VF progression, integrating macular GCIPL and parapapillary RNFL measurements is relevant to facilitate early detection of disease deterioration in glaucoma patients., (Copyright © 2018 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Neutrophil dysfunction is strongly linked to type 2 diabetes mellitus (T2DM) pathophysiology, but the prognostic potential of neutrophil biomarkers remains largely unexplored due to arduous leukocyte isolation methods. Herein, a novel integrated microdevice is reported for single-step neutrophil sorting and phenotyping (chemotaxis and formation of neutrophil extracellular traps (NETosis)) using small blood volumes (fingerprick). Untouched neutrophils are purified on-chip from whole blood directly using biomimetic cell margination and affinity-based capture, and are exposed to preloaded chemoattractant or NETosis stimulant to initiate chemotaxis or NETosis, respectively. Device performance is first characterized using healthy and in vitro inflamed blood samples (tumor necrosis factor alpha, high glucose), followed by clinical risk stratification in a cohort of subjects with T2DM. Interestingly, "high-risk" T2DM patients characterized by severe chemotaxis impairment reveal significantly higher C-reactive protein levels and poor lipid metabolism characteristics as compared to "low-risk" subjects, and their neutrophil chemotaxis responses can be mitigated after in vitro metformin treatment. Overall, this unique and user-friendly microfluidics immune health profiling strategy can significantly aid the quantification of chemotaxis and NETosis in clinical settings, and be further translated into a tool for risk stratification and precision medicine methods in subjects with metabolic diseases such as T2DM., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Atherosclerosis, a chronic inflammatory disorder characterized by endothelial dysfunction and blood vessel narrowing, is the leading cause of cardiovascular diseases including heart attack and stroke. Herein, we present a novel tunable microfluidic atherosclerosis model to study vascular inflammation and leukocyte-endothelial interactions in 3D vessel stenosis. Flow and shear stress profiles were characterized in pneumatic-controlled stenosis conditions (0%, 50% and 80% constriction) using fluid simulation and experimental beads perfusion. Due to non-uniform fluid flow at the 3D stenosis, distinct monocyte (THP-1) adhesion patterns on inflamed [tumor necrosis factor-α (TNF-α) treated] endothelium were observed, and there was a differential endothelial expression of intercellular adhesion molecule-1 (ICAM-1) at the constriction region. Whole blood perfusion studies also showed increased leukocyte interactions (cell rolling and adherence) at the stenosis of healthy and inflamed endothelium, clearly highlighting the importance of vascular inflammation, flow disturbance, and vessel geometry in recapitulating atherogenic microenvironment. To demonstrate inflammatory risk assessment using leukocytes as functional biomarkers, we perfused whole blood samples into the developed microdevices (80% constriction) and observed significant dose-dependent effects of leukocyte adhesion in healthy and inflamed (TNF-α treated) blood samples. Taken together, the 3D stenosis chip facilitates quantitative study of hemodynamics and leukocyte-endothelial interactions, and can be further developed into a point-of-care blood profiling device for atherosclerosis and other vascular diseases.

Rapamycin is commonly used in chemotherapy and posttransplantation rejection suppression, where sustained release is preferred. Conventionally, rapamycin has to be administered in excess due to its poor solubility, and this often leads to cytotoxicity and undesirable side effects. In addition, rapamycin has been shown to be hydrolytically unstable, losing its bioactivity within a few hours. The use of drug delivery systems is hypothesized to preserve the bioactivity of rapamycin, while providing controlled release of this otherwise potent drug. This paper reports on the use of microparticles (MP) as a means to tune and sustain the delivery of bioactive rapamycin for up to 30 days. Rapamycin was encapsulated (100% efficiency) in poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), or a mixture of both via an emulsion method. The use of different polymer types and mixture was shown to achieve a variety of release kinetics and profile. Released rapamycin was subsequently evaluated against breast cancer cell (MCF-7) and human lymphocyte cell (Jurkat). Inhibition of cell proliferation was in good agreement with in vitro release profiles, which confirmed the intact bioactivity of rapamycin. For Jurkat cells, the suppression of cell growth was proven to be effective up to 20 days, a duration significantly longer than free rapamycin. Taken together, these results demonstrate the ability to tune, sustain, and preserve the bioactivity of rapamycin using MP formulations. The sustained delivery of rapamycin could lead to better therapeutic effects than bolus dosage, at the same time improving patient compliance due to its long-acting duration.

Host defence peptides (HDPs) derived from the C-terminus of thrombin are proteolytically generated by enzymes released during inflammation and wounding. In this work, we studied the effects of the prototypic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), on neutrophil functions. In vitro, GKY25 was shown to decrease LPS-induced neutrophil activation. In addition, the peptide induced CD62L shedding on neutrophils without inducing their activation. Correspondingly, GKY25-treated neutrophils showed reduced attachment and rolling behaviour on surfaces coated with the CD62L ligand E-selectin. The GKY25-treated neutrophils also displayed a dampened chemotactic response against the chemokine IL-8. Furthermore, in vivo, mice treated with GKY25 exhibited a reduced local ROS response against LPS. Taken together, our results show that GKY25 can modulate neutrophil functions in vitro and in vivo.

Vessel geometries in microengineered in vitro vascular models are important to recapitulate a pathophysiological microenvironment for the study of flow-induced endothelial dysfunction and inflammation in cardiovascular diseases. Herein, we present a simple and novel extracellular matrix (ECM) hydrogel patterning method to create perfusable vascularized microchannels of different geometries based on the concept of capillary burst valve (CBV). No surface modification is necessary and the method is suitable for different ECM types including collagen, matrigel and fibrin. We first created collagen-patterned, endothelialized microchannels to study barrier permeability and neutrophil transendothelial migration, followed by the development of a biomimetic 3D endothelial-smooth muscle cell (EC-SMC) vascular model. We observed a significant decrease in barrier permeability in the co-culture model during inflammation, which indicates the importance of perivascular cells in ECM remodeling. Finally, we engineered collagen-patterned constricted vascular microchannels to mimic stenosis in atherosclerosis. Whole blood was perfused (1-10 dyne cm -2 ) into the microdevices and distinct platelet and leukocyte adherence patterns were observed due to increased shear stresses at the constriction, and an additional convective flow through the collagen. Taken together, the developed hydrogel patterning technique enables the formation of unique pathophysiological architectures in organ-on-chip microsystems for real-time study of hemodynamics and cellular interactions in cardiovascular diseases.